Methods for restoring wild-type p53 gene function

ABSTRACT

Methods are provided for restoring wild-type p53 gene function to a cell. Such methods include gene therapy. Typically, this will stop tumor cells from proliferating.

This application is a divisional of Ser. No. 08/035,366, filed on Mar.22, 1993, which is a continuation-in-part of Ser. No. 07/860,758, filedon Mar. 31, 1992, now U.S. Pat. No. 5,362,623, which is acontinuation-in-part of Ser. No. 07/715,182, filed Jun. 14, 1991, nowabandoned, as well as a continuation-in-part of Ser. No. 07/928,661,filed Aug. 17, 1992, now abandoned, which is a continuation of Ser. No.07/446,584, filed Dec. 6, 1989, now abandoned, which is acontinuation-in-part of Ser. No. 07/330,566, filed Mar. 29, 1989, nowabandoned.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant numbersGM07309, GM07184, HD20619, CA42857, CA28854, CA47527, CA35494, NS23427and CA43460 awarded by the National Institutes of Health.

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of cancer diagnostics andtherapeutics. More particularly, the invention relates to detection andremediation of the loss and or alteration of wild-type p53 genes fromtumor tissues.

BACKGROUND OF THE INVENTION

Recent studies have elucidated several genetic alterations that occurduring the development of colorectal tumors, the most common of whichare deletions of the short arm of chromosome 17 (17p). While somegenetic alterations such as RAS gene mutations, appear to occurrelatively early during colorectal tumor development, chromosome 17pdeletions are often late events associated with the transition from thebenign (adenomatous) to the malignant (carcinomatous) state. SeeVogelstein et al., New England Journal of Medicine, Vol. 319, p525,1988.

Because carcinomas are often lethal, while the precursor adenomas areuniformly curable, the delineation of the molecular events mediatingthis transition are of considerable importance. The occurrence ofallelic deletions of chromosome 17p in a wide variety of cancers besidesthose of the colon, including those of the breast and lung, furtheremphasizes the importance of genes residing on chromosome 17p in theneoplastic process. Because allelic deletions have been reported toencompass a large area of chromosome 17p, there is a need in the art fordefining the particular genetic region which is responsible for theneoplastic progression.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordiagnosing a neoplastic tissue of a human.

It is another object of the invention to provide a method of supplyingwild-type p53 gene function to a cell which has lost said gene function.

It is yet another object of the invention to provide a kit fordetermination of the nucleotide sequence of the p53 gene by using thepolymerase chain reaction.

It still another object of the invention to provide a nucleic acid probefor detection of mutations in the human p53 gene.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment of thepresent invention a method of diagnosing a neoplastic tissue of a humanis provided comprising: isolating from a human a tissue suspected ofbeing neoplastic; and detecting loss of wild-type p53 genes or theirexpression products from said tissue, said loss indicating neoplasia ofthe tissue.

In another embodiment of the present invention a method is provided forsupplying wild-type p53 gene function to a cell which has lost said genefunction by virtue of a mutation in the p53 gene, comprising:introducing a wild-type p53 gene into a cell which has lost said genefunction such that said wild-type gene is expressed in the cell.

In yet another embodiment a kit is provided for determination of thenucleotide sequence of the p53 gene by polymerase chain reaction. Thekit comprises: a set of pairs of single stranded DNA primers, said setallowing synthesis of all nucleotides of the p53 gene coding sequences.

In still another embodiment of the invention a nucleic acid probe isprovided which is complementary to human wild-type p53 gene sequencesand which can form mismatches with mutant p53 genes, thereby allowingtheir detection by enzymatic or chemical cleavage or by shifts inelectrophoretic mobility.

The present invention provides the art with the information that the p53gene is, in fact, the target of both deletional and point mutationalalterations on chromosome 17p which are associated with the process oftumorigenesis. This information allows highly specific assays to be doneto assess the neoplastic status of a particular tumor tissue as well asthe performance of therapeutic anti-cancer methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the analysis of allelic losses on chromosome 17p inthe human tissue of two patients, S51 and S103.

FIG. 2 shows a map of the common region of deletions on chromosome 17pin colorectal tumors. The chromosomal positions of 20 restrictionfragment length polymorphism (RFLP) markers from chromosome 17p areindicated. The markers were previously mapped to seven sub-chromosomalregions (indicated A to F). Hybridization results for eight tumors areshown on the right, with patient identification numbers indicated at thebottom. A filled circle indicates loss of one parental allele in thetumor; a cross-hatched circle indicates retention of both parentalalleles; an open circle indicates that the marker was not informative,i.e. the patient's normal tissue was not heterozygous for the marker.The premise of the composite pattern is that there is a single targetgene on 17p. Therefore, markers for which heterozygosity was retained inany of the eight tumors (i.e., cross-hatched circles) would be outsidethe target locus.

FIG. 3 shows a Northern blot analysis of p53 mRNA in colorectal tumors.The RNA in lanes 1-6 and 12 was prepared from human tissues (normalcolonic mucosa (N) or carcinoma biopsies (C)). The RNA in lanes 7-11 and13 was prepared from colorectal carcinoma cell lines.

FIG. 4 shows analysis of the products of polymerase chain amplificationof a 111 bp fragment surrounding the p53 gene codon 143. Lanes 1,2:colorectal tumor xenograft Cx1; lanes 3,4: normal fibroblasts from thepatient providing Cx1; lanes 5,6: colorectal tumor xenograft Cx3; lanes7,8: normal fibroblasts from the patient providing Cx3.

FIG. 5 shows polymerase chain reaction analysis of p53 codon 175. Lanes1,2: colorectal tumor xenograft Cx1; lanes 3,4: normal fibroblasts fromthe patient providing Cx1; lanes 5,6: colorectal tumor xenograft Cx3.Samples in even numbered lanes only were digested with Hha I.

FIG. 6 depicts RNase protection analysis of p53 mRNA. Cellular RNA washybridized with radiolabeled anti-sense p53 RNA probe, and the hybridsdigested with RNase A. The RNA was derived from: lane 1: S115, carcinomabiopsy; lane 2: SW1417; lane 3: SW948; lane 4: RKO; lane 5: SW480; lane6: RCA; lane 7: GEO; lane 8 FET; lane 9: xenograft Cx3; lane 10: normalcolonic mucosa; lane 11: yeast tRNA; lane 12: probe alone (not RNase Adigested); lane 13: SW1417 (long exposure). The fragments marked witharrowheads in lanes 5,6, and 13 were not present in the other samples.

FIG. 7 diagrams the strategies used for amplification of p53 genesequences. mRNA was used to generate a cDNA template for a polymerasechain reaction (PCR) employing primers P1 and P2 (top). The PCR productwas 1.3 kb and included the entire coding region. Alternatively, totalgenomic DNA was used in a PCR reaction employing primers P3 and P4. ThePCR product was 2.9 kb and included exons 4-9 (bottom). The numberedboxes indicate exons and the vertical dotted lines indicate the start(ATG) and stop (TGA) codons respectively.

FIG. 8 shows examples of sequencing reactions demonstrating p53 genemutations. The templates used for the sequencing reactions shown inpanels 1-4 consisted of pools of greater than 103 clones generated fromPCR products. Tumor #13 genomic DNA contained a mutation at codon 239(antisense GCT, Panel 2), instead of the wild type sequence (GTT) foundin the genomic DNA from normal lymphocytes from the same patient (Panel1). Panel 4 shows a sequencing reaction of pooled cDNA clones from tumor#16 showing that both wild-type codon 281 (GAC) and mutant codon 281(GGC) were both expressed. Only the wild type sequence (GAC) was foundin pooled genomic DNA clones from normal lymphocytes of this patient(Panel 3).

FIG. 9 maps the p53 point mutations involved in human cancer. Each ofthe missense mutations listed in Table 1 is indicated with an arrow. Inaddition, the two point mutations described previously (Baker, et al.,Science, vol. 244, p. 217, 1989) in human cancers (at codons 143 and175) are also included. The four regions containing most (86%) of themutations are indicated by the black bars marked A-D.

FIG. 10A shows RNase protection analysis of transfected clonal lines. Alabeled antisense p53 probe was hybridized with total cellular RNA, anddigested with RNase A. Endogenous RNA included all sequences representedin the labeled probe. Exogenous p53 RNA produced from the expressionvectors extended only about ⅔ of the length of the probe.

FIG. 10B shows Southern blot analysis of transfected clonal lines. Theexogenous p53 gene was present on a 1.8 kb BamHI fragment. Theendogenous p53 gene gave rise to a 7.8 kb BamHI fragment. Other sizedfragments presumably arose by rearrangements.

FIG. 11A shows expression analysis of pooled clones; the analysis was asdescribed in FIG. 10.

FIG. 11B shows a Southern blot analysis of SW480 pooled clones.

DETAILED DESCRIPTION

It is a discovery of the present invention that mutational eventsassociated with tumorigenesis occur in the p53 gene on chromosome 17p.Although it was previously known that deletion of alleles on chromosome17p were common in certain types of cancers, it was not known that thedeletions shared a common region which includes the p53 gene. Further itwas not known that a second mutational event on the sister chromosome ofthat carrying the deletions was also affected by mutation in the p53gene. The mutation of the sister chromosome does not involve grossrearrangements such as deletions, insertions or inversions, but ratherpoint mutations located in a variety of positions throughout the p53gene. Although the inventor does not wish to be bound by the followingtheory, it is proposed as a possible mechanism which explains theobserved results. It is believed that the point mutation occurs firstand the deletion event occurs second, as the latter event is correlatedwith the change of a tumor from an adenomatous to a carcinomatous state.

According to the diagnostic method of the present invention, loss of thewild-type p53 gene is detected. The loss may be due to either deletionaland/or point mutational events. If only a single p53 allele is mutated,an early neoplastic state is indicated. However, if both alleles aremutated then a late neoplastic state is indicated. The p53 allele whichis not deleted (i.e., that on the sister chromosome to the chromosomecarrying the deletion) can be screened for point mutations, such asmissense, and frameshift mutations. Both of these types of mutationswould lead to non-functional p53 gene products. In addition, pointmutational events may occur in regulatory regions, such as in thepromoter of the p53 gene, leading to loss or diminution of expression ofthe p53 mRNA.

In order to detect the loss of the p53 wild-type gene in a tissue, it ishelpful to isolate the tissue free from surrounding normal tissues.Means for enriching a tissue preparation for tumor cells are known inthe art. For example, the tissue may be isolated from paraffin orcryostat sections. Cancer cells may also be separated from normal cellsby flow cytometry. These as well as other techniques for separatingtumor from normal cells are well known in the art. If the tumor tissueis highly contaminated with normal cells, detection of mutations is moredifficult.

Detection of point mutations may be accomplished by molecular cloning ofthe p53 allele (or alleles) present in the tumor tissue and sequencingthat allele(s) using techniques well known in the art. Alternatively,the polymerase chain reaction can be used to amplify p53 gene sequencesdirectly from a genomic DNA preparation from the tumor tissue. The DNAsequence of the amplified sequences can then be determined. Thepolymerase chain reaction itself is well known in the art. See e.g.,Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. Pat. No. 4,683,202;and U.S. Pat. No. 4,683,195. Specific primers which can be used in orderto amplify the p53 gene will be discussed in more detail below.

Specific deletions of p53 genes can also be detected. For example,restriction fragment length polymorphism (RFLP) probes for the p53 geneor surrounding marker genes can be used to score loss of a p53 allele.Other techniques for detecting deletions, as are known in the art can beused.

Loss of wild-type p53 genes may also be detected on the basis of theloss of a wild-type expression product of the p53 gene. Such expressionproducts include both the mRNA as well as the p53 protein productitself. Point mutations may be detected by sequencing the mRNA directlyor via molecular cloning of cDNA made from the mRNA. The sequence of thecloned cDNA can be determined using DNA sequencing techniques which arewell known in the art. The cDNA can also be sequenced via the polymerasechain reaction (PCR) which will be discussed in more detail below.

Alternatively, mismatch detection can be used to detect point mutationsin the p53 gene or its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof tumors. An example of a mismatch cleavage technique is the RNaseprotection method, which is described in detail in Winter et al., Proc.Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science,Vol. 230, p. 1242, 1985. In the practice of the present invention themethod involves the use of a labeled riboprobe which is complementary tothe human wild-type p53 gene. The riboprobe and either mRNA or DNAisolated from the tumor tissue are annealed (hybridized) together andsubsequently digested with the enzyme RNase A which is able to detectsome mismatches in a duplex RNA structure. If a mismatch is detected byRNase A, it cleaves at the site of the mismatch. Thus, when the annealedRNA preparation is separated on an electrophoretic gel matrix, if amismatch has been detected and cleaved by RNase A, an RNA product willbe seen which is smaller than the full-length duplex RNA for theriboprobe and the p53 mRNA or DNA. The riboprobe need not be the fulllength of the p53 mRNA or gene but can be a segment of either. If theriboprobe comprises only a segment of the p53 mRNA or gene it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl.Acad. Sci. USA, vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad.Sci. USA, vol. 72, p. 989, 1975. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR (see below) before hybridization.

DNA sequences of the p53 gene from the tumor tissue which have beenamplified by use of polymerase chain reaction may also be screened usingallele-specific probes. These probes are nucleic acid oligomers, each ofwhich contains a region of the p53 gene sequence harboring a knownmutation. For example, one oligomer may be about 30 nucleotides inlength, corresponding to a portion of the p53 gene sequence. At theposition coding for the 175th codon of p53 gene the oligomer encodes analanine, rather than the wild-type codon valine. By use of a battery ofsuch allele-specific probes, the PCR amplification products can bescreened to identify the presence of a previously identified mutation inthe p53 gene. Hybridization of allele-specific probes with amplified p53sequences can be performed, for example, on a nylon filter.Hybridization to a particular probe indicates the presence of the samemutation in the tumor tissue as in the allele-specific probe.

Loss of wild-type p53 genes can also be detected by screening for lossof wild-type p53 protein function. Although all of the functions whichthe p53 protein undoubtedly possesses have yet to be elucidated, atleast two specific functions are known. Protein p53 binds to the SV40large T antigen as well as to the adenovirus E1B antigen. Loss of theability of the p53 protein to bind to either or both of these antigensindicates a mutational alteration in the protein which reflects amutational alteration of the gene itself. Alternatively, a panel ofmonoclonal antibodies could be used in which each of the epitopesinvolved in p53 functions are represented by a monoclonal antibody. Lossor perturbation of binding of a monoclonal antibody in the panel wouldindicate mutational alteration of the p53 protein and thus of the p53gene itself. Any means for detecting an altered p53 protein can be usedto detect loss of wild-type p53 genes.

Mutant p53 genes or gene products can also be detected in body samples,such as, serum, stool, or other body fluids, such as urine and sputum.The same techniques discussed above for detection of mutant p53 genes orgene products in tissues can be applied to other body samples. Byscreening such body samples, a simple early diagnosis can be achievedfor many types of cancers. In addition, the progress of chemotherapy orradiotherapy can be monitored more easily by testing such body samplesfor mutant p53 genes or gene products.

The method of the present invention for diagnosis of neoplastic tissueis applicable across a broad range of tumors. These include lung,breast, brain, colorectal, bladder, mesenchyme, prostate, liver as wellas stomach tumors. In addition the method may be used in leukemias andosteosarcomas. It thus appears that the p53 gene has a role in thedevelopment of a broad range of tumors. The methods of diagnosis of thepresent invention are applicable to any tumor in which p53 has a role intumorigenesis. The diagnostic method of the present invention is usefulfor clinicians so that they can decide upon an appropriate course oftreatment. For example, a tumor displaying loss of both p53 allelessuggests a more aggressive therapeutic regimen than a tumor displayingloss of only one p53 allele.

The kit of the present invention is useful for determination of thenucleotide sequence of the p53 gene using the polymerase chain reaction.The kit comprises a set of pairs of single stranded DNA primers whichcan be annealed to sequences within or surrounding the p53 gene in orderto prime amplifying DNA synthesis of the p53 gene itself. The completeset allows synthesis of all of the nucleotides of the p53 gene codingsequences. The set of primers may or may not allow synthesis of bothintron and exon sequences. However, it should allow synthesis of allexon sequences.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme sites appended to their 5′ ends.Thus, all nucleotides of the primers are derived from p53 sequences orsequences adjacent to p53 except the few nucleotides necessary to form arestriction enzyme site. Such enzymes and sites are well known in theart. The primers themselves can be synthesized using techniques whichare well known in the art. Generally, the primers can be made usingsynthesizing machines which are commercially available.

In a preferred embodiment, the set of primer pairs comprises five primerpairs which are listed below. Primer pair 1:5′-GGAATTCCACGACGGTGACACG-3′ (SEQ ID NO:1) and5′-GGAATTCGGTGTAGGAGCTGCTGG-3′ (SEQ ID NO:2); pair 2:5′-GGAATTCCCAGAATGCCAGAGGC-3′ (SEQ ID NO:3);5′-GGAATTCATGTGCTGTGACTGCTTG-3′ (SEQ ID NO:4); pair 3:5′-GGAATTCCACACCCCCGCCCG-3′ (SEQ ID NO:5) and5′-GGAATTCATGCCGCCCATGCAG-3′ (SEQ ID NO:6); pair 4:5′-GGAATTCTGACTGTACCACCATCC-3′ (SEQ ID NO:7)and5′-GGAATTCTCCATCCAGTGGTTTC-3′ (SEQ ID NO:8) ; pair 5:5′-GGAATTCCCAACAACACCAGCTCC-3′ (SEQ ID NO:9) and5′-GGAATTCAAAATGGCAGGGGAGGG-3′ (SEQ ID NO:10).

The nucleic acid probes provided by the present invention are useful inthe RNase protection method for detecting point mutations alreadydiscussed above. They may also be used to detect mismatches with the p53gene or mRNA using other techniques. Mismatches can be detected usingother enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamine orosmium tetroxide and piperidine), or changes in electrophoretic mobilityof mismatched hybrids as compared to totally matched hybrids. Thesetechniques are known in the art. See, Cotton, supra, Shenk, supra,Myers, supra, Winter, supra, and Novack, et al., Proc. Natl. Acad. Sci.USA, vol. 83, p. 586, 1986. If a riboprobe is used to detect mismatcheswith mRNA, it is complementary to the mRNA of the human wild-type p53gene. The riboprobe thus is an anti-sense probe in that it does not codefor the p53 protein because it is of the opposite polarity to the sensestrand. The riboprobe generally will be radioactively labeled which canbe accomplished by any means known in the art. If the riboprobe is usedto detect mismatches with DNA it can be of either polarity, sense oranti-sense. Similarly, DNA probes also may be used to detect mismatches.Probes may also be complementary to mutant alleles of p53 gene. Theseare useful to detect similar mutations in other patients on the basis ofhybridization rather than mismatches. These are discussed above andreferred to as allele-specific probes.

According to the present invention a method is also provided ofsupplying wild-type p53 function to a cell which carries mutant p53alleles. The wild-type p53 gene or a part of the gene may be introducedinto the cell in a vector such that the gene remains extrachromosomal.In such a situation the gene will be expressed by the cell from theextrachromosomal location. If the mutant p53 genes present in the cellare expressed, then the wild-type p53 gene or gene portion should beexpressed to a higher level than that of the mutant gene. This isbecause the mutant forms of the protein are thought to oligomerize withwild-type forms of the protein. (Eliyahu et al., Oncogene, Vol. 3, p.313, 1988.) If a gene portion is introduced and expressed in a cellcarrying a mutant p53 allele, the gene portion should encode a part ofthe p53 protein which is required for non-neoplastic growth of the cell.More preferred is the situation where the wild-type p53 gene or a partof it is introduced into the mutant cell in such a way that itrecombines with the endogenous mutant p53 gene present in the cell. Suchrecombination would require a double recombination event which wouldresult in the correction of the p53 gene mutation. Vectors forintroduction of genes both for recombination and for extrachromosomalmaintenance are known in the art and any suitable vector may be used.

According to another embodiment of the invention viral vectors can beused to introduce wild-type p53 alleles to cells. Such vectors aredesigned to express non-viral genes. Classes of such vectors includewithout limitation: retroviral vectors, adenoviral vectors, herpes virusvectors and adeno-associated virus vectors. These vectors can integrateinto the genome and even after integration direct the expression ofinserted genes, such as p53. Plasmid expression vectors can also beused. These may integrate into the genome or replicate autonomously,while directing the expression of p53.

Polypeptides or other molecules which have p53 activity may be suppliedto cells which carry mutant p53 alleles. The active molecules can beintroduced into the cells by microinjection or by liposomes, forexample. Alternatively, some such active molecules may be taken up bythe cells, actively or by diffusion. Supply of such active moleculeswill effect an earlier neoplastic state.

Predisposition to cancers can be ascertained by testing normal tissuesof humans. For example, a person who has inherited a germline p53mutation would be prone to develop cancers. This can be determined bytesting DNA from any tissue of the person's body. Most simply, blood canbe drawn and DNA extracted from the cells of the blood. Loss of awild-type p53 allele, either by point mutation or by deletion, can bedetected by any of the means discussed above. DNA can also be extractedand tested from fetal tissues for this purpose.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention which has been described inbroad terms above.

EXAMPLES Example 1

This example demonstrates that the deletions found on chromosome 17p inhuman colorectal carcinomas share a common region between bands 17p12and 17p13.3.

Twenty DNA probes detecting restrictions fragment length polymorphisms(RFLPs) on chromosome 17p were used to examine the patterns of alleliclosses in colorectal tumors. These probes have been mapped to sevendiscrete regions of 17p on the basis of their hybridization tohuman-rodent somatic cell hybrids containing parts of chromosome 17p (P.van Tuinen, D. C. Rich, K. M. Summers, D. H. Ledbetter, Genomics 1, 374(1987); P. van Tuinen et al., Am. J. Hum. Gen. 43, 587 (1988); P. R.Fain et al., Genomics 1, 340 (1987); unpublished data of D. H. Ledbetterand D. F. Barker).

DNA was obtained from 58 carcinoma specimens and compared to DNA fromadjacent normal colonic mucosa. Allelic losses were scored if either ofthe two alleles present in the normal cells was absent in the DNA fromthe tumor cells. Allelic deletions can be difficult to detect in DNAprepared from whole tumors because most solid tumors contain asignificant number of non-neoplastic stromal and inflammatory cells. Forthis reason, regions of tumors containing a high proportion ofneoplastic cells were identified histopathologically and isolated, andDNA was prepared from cryostat sections of these regions as describedpreviously (S. Goelz, S. R. Hamilton, B. Vogelstein, Biochem. Biophys.Res. Commun. 130, 118 (1985); E. R. Fearon, A. Feinberg, S. R. Hamilton,B. Vogelstein, Nature 318, 377 (1985). Grossly normal colonic mucosaadjacent to the tumors was obtained from each patient and used toprepare control DNA.

The two parental alleles could be distinguished in the normal mucosa ofeach patient with at least 5 of the 20 RFLP markers (the “informative”markers for each case). Seventy-seven percent of the tumors exhibitedallelic losses of at least 3 markers. Studies of 8 tumors which retainedheterozygosity for some but not all markers on chromosome 17p enabledthe definition of a common region of deletion.

FIG. 1 shows a sample of the data collected from two patients. DNA fromnormal (N) and carcinoma (C) tissue of patients S51 and S103 wasdigested with restriction endonucleases and the fragments separated byelectrophoresis. After transfer to nylon filters, the DNA was hybridizedto radiolabeled probes. Techniques used for DNA purification restrictionendonuclease digestion, electrophoresis, transfer and hybridization wereperformed as described (B. Vogelstein et al., N. Engl. J. of Med. 319,525 (1988); Goelz, supra; Fearon, supra.) TaqI digestion was used forpanels A, B, C, and F, BamHI for panel D and Mspl for panel E.Autoradiographs of the washed filters are shown. The alleles designated“1” and “2” refer to the larger and smaller polymorphic alleles,respectively, present in the normal DNA samples. The probes used were:A: MCT35.2; B: EW301; C: YNH137.3; D: YNZ22.1; E: MCT.35.1; F: EW505.Deletions of allele 1 can be seen in panels A and E; deletions of allele2 in panels B and D.

The tumor from patient S51 had retained both parental alleles of threemarkers from the distal region of 17p, but had lost one of all moreproximal markers that were formative (FIGS. 1, A-C). This implied thatthe target of the allelic loss in this tumor was proximal to the threeretained markers. Analysis of the pattern of marker loss is shown inFIG. 2. The tumor from patient S103 had retained both parental allelesat all informative loci proximal to EW505, but had allelic deletions ofseveral more distal markers (FIGS. 1, D-F). The combined data depictedin FIG. 2 indicated that the smallest common region of deletion extendedbetween markers within band 17p12 to those within band 17p13.3. Thislocalization is based on the assumption that the same 17p locus was thetarget of deletion in all of the tumors.

Example 2

This example demonstrates that the non-deleted p53 alleles in colorectalcarcinomas carrying a p53 deletion are not rearranged.

First, p53 cDNA probes detecting exons spread over 20,000 base pairs(including all protein encoding exons) [P. Lamb, L. V. Crawford, Mol.Cell. Biol. 6, 1379 (1986); R. Zakut-Houri, B. Bienz-Tadmor, D. Givol,M. Oren, EMBO J. 4, 1251 (1985); N. Harris E. Brill, O. Shahat, M.Prokocimer, T. E. Admas, Mol. Cell. Biol., 6, 4650 (1986); G.Matlashewski et al., Molec. Cell. Biol. 7, 961 (1987); V. L. Buchman etal., Gene 70, 245 (1988)] were used to examine the DNA of 82 colorectalcarcinomas (50 primary specimens and 32 cell lines) in Southern blottingexperiments.

No rearrangements of the p53 gene were observed with EcoRI or BamHIdigests, nor were deletions of both alleles seen. Because p53 expressionmight be affected by gross genetic alterations further removed from p53coding sequences, pulsed-field gel electrophoresis was used to examinelarge restriction fragments encompassing the p53 gene. The restrictionendonucleases EcoRV, PaeR7 I, NotI, and SalI generated p53gene-containing fragments of 45-350 kb from the DNA of normal cells. Noalterations were detected in the DNA from any of 21 colorectal tumorcell lines examined with each of these four enzymes.

Example 3

This example demonstrates that the non-deleted p53 alleles in colorectalcarcinomas carrying a p53 deletion express mRNA of the normal size andin most cases normal amounts.

Northern blot experiments were performed on RNA from 22 colorectaltumors (6 primary tumors and 16 cell lines). Because p53 expression hasbeen correlated with cellular growth and/or transformation other geneswhose expression is similarly regulated were used as controls (c-myc,histone H3, and phosphoglycerate kinase).

RNA was purified from grossly normal colonic mucosa, primary carcinomaspecimens or tumor cell lines, and separated by electrophoresis. Celllines were generously provided by D. and M. Brattain or obtained fromthe American Type Culture Collection, Rockville, Maryland. Totalcellular RNA was isolated by the acid-guanidinium extraction method (P.Chomczynski, N. Sacchi, Anal. Biochem. 162, 156 (1987)). Five microgramswere separated by electrophoresis through a 1.5% 2(N-morpholino) ethanesulfonic acid-formaldehyde agarose gel and electrophoreticallytransferred to nylon filters. The RNA was transferred to nylon filtersand hybridized with a radiolabeled p53 gene probe. Labelling of theprobes, hybridization, washing and autoradiography were performed asdescribed. (Fearon et al., Science, Vol. 238, p. 193, 1987; Vogelsteinet al., N. Engl. J. of Med., Vol. 319, p. 525, 1988; and Goelz, supra;and Fearon, Nature, supra . Autoradiographs were exposed for 18-24hours.

The p53 probe was a 1.8 kb Xbal fragment of a p53 cDNA clone generouslyprovided by D. Givol (EMBO J., vol. 4, p. 1251 (1985)). The c-myc probewas a 1.6 kb genomic Sstl fragment containing exon 2 of c-myc (K.Alitalo et al., Proc. Nat'l. Acad. Sci. USA 80, 1707 (1983)). Thesignals were removed from the filter, and the blot was re-hybridizedwith a c-myc gene probe. Autoradiographs of the hybridized filters areshown in FIG. 3. The size of the p53 mRNA detected was 2.8 kb, and thesize of the c-myc mRNA was 2.5 kb.

The RNA in lanes 1-6 and lane 12 was prepared from human tissues (normalcolonic mucosa (N) or carcinoma biopsies (C)). The RNA in lanes 7-11 and13 was prepared from colorectal carcinoma cell lines. Lanes 1, 2:Patient S345, N and C, respectively. Lanes 3, 4: Patient S353, N and C,respectively. Lanes 5, 6, Patient S369, N and C, respectively. Lane 7:SW837, Lane 8: SW480, Lane 9: LoVo, Lane 10: SW948, Lane 11: SW1417,Lane 12: Patient S115, C, Lane 13: RKO.

The size of p53 mRNA was normal (2.8 kb) in all 22 tumors. Moreover, therelative abundance of p53 gene mRNA was usually at least as great incolorectal tumor cells as in normal colonic mucosa confirming theresults of Calabretta et al. (Cancer Research, Vol. 46, p. 738 (1986)).However, in four tumors, (lanes 10-13) relatively little expression ofp53 mRNA was observed compared to that in the other tumors. This lowlevel of expression of p53 was specific in that c-myc, histone H3, andphosphoglycerate kinase mRNAs were expressed in these four tumors atlevels similar to those seen in other colorectal tumors and at least ashigh as in non-neoplastic colonic mucosa.

Example 4

This example demonstrates that the non-deleted p53 allele in a primarytumor carries a point mutation at codon 143.

A tumor was chosen which had an allelic deletion of chromosome 17p yetexpressed significant quantities of p53 mRNA. A cDNA clone originatingfrom the remaining p53 allele was isolated and sequenced to determinewhether the gene product was abnormal.

For practical reasons, a nude mouse xenograft (Cx3) of a primary tumorwas selected for this test. Primary tumors contain non-neoplastic cellswhich could contribute p53 mRNA, while in xenografts the non-neoplasticcells (derived from the mouse) could not be the source of a human p53cDNA clone. Cx3, like over 75% of colorectal carcinomas, had allelicdeletions of several RFLP (restriction fragment length polymorphism)markers on chromosome 17 and expressed significant amounts of p53 mRNA.

A nearly full-length p53 cDNA was cloned from Cx3 mRNA using standardtechniques. Double stranded cDNA was synthesized as described by U.Gubler and B. J. Hoffman, Gene 25 263 (1983) and cloned into the lambdagt10 vector. The cDNA insert was subcloned into Bluescript KS(Stratagene Cloning Systems, LaJolla, Calif.) and nested deletions weremade with exonuclease III (S. Henekoff, Gene 28, 351 [1984]). Sequenceswere obtained from double-stranded templates using modified T7polymerase as described by S. Tabor and C. C. Richardson, Proc. Nat'l.Acad. Sci. USA 84, 4767 (1987) and R. Kraft, J. Tardiff, K. S. Krauterand I. A. Leinwand, Biotechniques 6, 544 (1988).

The clone extended 2567 nucleotides from position −198 relative to thetranslation initiation site to the polyadenosine tail. The clone wassequenced by the dideoxy chain-termination method and one nucleotidedifference was identified in comparison with published p53 cDNAsequences (See, Lamb, supra; Zakut-Houri, supra; Harris, supra;Matlashewski; supra; and Buchman, supra . A transition from T to C hadoccurred within codon 143 (GTG to GCG), resulting in a change of theencoded amino acid from valine to alanine.

To ensure that the sequence change was not an artifact of cDNA cloning,the polymerase chain reaction [PCR, (Saiki, et al., Science, Vol. 239,p. 487, 1988)] was used to amplify a 111 base pair (bp) sequencesurrounding the presumptive mutation from genomic DNA of Cx3.

DNA was incubated in the presence of Taq polymerase with primeroligomers complementary to sequences 68 base pairs upstream and 43 basepairs downstream of codon 143. The upstream primer used was5′-TTCCTCTTCCTGCAGTACTCC-3′ (SEQ ID NO:11); all but 6 nucleotides ofthis primer were derived from the p53 intron 4 sequence determined byBuchman et al., supra. The downstream primer was5′-GACGCGGGTGCCGGGCGG-3′ (SEQ ID NO:12). After 35 cycles of denaturation(one minute, 93°), annealing (2 minutes, 55°) and elongation (2 minutes,70°) amplified DNA fragments of 111 bp were generated. Followingelectrophoresis, the 111 bp amplified fragments were eluted from apolyacrylamide gel and purified by extraction with phenol andchloroform.

Analysis of the PCR product was facilitated by the observation that thepresumptive mutation created a new HhaI site (GCGC at nt 427-430). Analiquot of each of the purified DNA fragments was digested with HhaI,separated by electrophoresis on a non-denaturing polyacrylamide gel, andelectrophoretically transferred to nylon filters. The fragments werehybridized with a radioactive p53 probe generated from a 1.8 kb XbaIfragment of a p53 cDNA clone provided by D. Givol (Zakot-Houri, supra .

The 111 bp PCR product from tumor Cx3 was cleaved with HhaI to producethe expected 68 and 43 subfragments (FIG. 4, lanes 5 and 6). The 111 bpPCR product from the DNA of normal cells of the patient providing Cx3was not cleaved with HhaI (lanes 7 and 8), nor were the PCR products of37 other DNA samples prepared from the normal tissues, primarycolorectal tumors, or xenografts of other patients (examples in FIG. 4,lanes 1-4). Therefore, the valine to alanine substitution present inthis tumor was the result of a specific point mutation not present inthe germline of the patient.

A small amount of a contaminating 73 base pair PCR product was presentin most of the eluates; the contaminant was not cleaved by HhaI,however, so that it did not interfere with the analysis.

Example 5

Colorectal carcinoma xenograft Cx1, like Cx3, had allelic deletions ofseveral markers on chromosome 17p and expressed considerable amounts ofnormal size p53 mRNA. First strand cDNA was generated from Cx1 RNA usingrandom hexamers in the presence of reverse transcriptase (E. Noonan andI. B. Roninson, Nucleic Acids Research 16, 10366 [1988]). This cDNA wasused in five separate PCR reactions to generate fragments correspondingto nucleotides −59 to 246 (primer pair 1), 189 to 508 (primer pair 2),443 to 740 (primer pair 3), 679 to 979 (primer pair 4), and 925 to 1248(primer pair 5). These fragments contained all coding sequences of thep53 gene. Primer pair 1: 5′-GGAATTCCACGACGGTGACACG-3′ (SEQ ID NO: 1) and5′-GGAATTCGGTGTAGGAGCTGCTGG-3′ (SEQ ID NO:2); pair 2:5′-GGAATTCCCAGAATGCCAGAGGC-3′ (SEQ ID NO:3) and5′-GGAATTCATGTGCTGTGACTGCTTG-3′ (SEQ ID NO:4); pair 3:5′-GGAATTCCACACCCCCGCCCG-3′ (SEQ ID NO:5) and5′-GGAATTCATGCCGCCCATGCAG-3′ (SEQ ID NO:6); pair 4:5′-GGAATTCTGACTGTACCACCATCC-3′ (SEQ ID NO:7) and5′-GGAATTCTCCATCCAGTGGTTTC-3′ (SEQ ID NO:8); pair 5:5′-GGAATTCCCAACAACACCAGCTCC-3′ (SEQ ID NO:9) and5′-GGAATTCAAAATGGCAGGGGAGGG-3′ (SEQ ID NO:10). All primers hadextraneous nucleotides comprising EcoRI cleavage sites at their 5′ endsto facilitate cloning. The PCR products were cloned in the EcoRI site ofBluescript SK and sequenced as described in Example 4. Only 1 base pairchange was identified (transition from CGC to CAC) and this change atcodon 175 was found in two independent clones.

To ensure that the sequence change represented a mutation rather than asequence polymorphism, PCR was used to amplify a fragment containingcodon 175 from the genomic DNA of tumor Cx1 and normal cells. PCR wasused to amplify a 319 bp fragment containing intron 5 and surroundingexon sequences. The upstream primer was the same as used for primer pair3 and the downstream primer was 5′-CGGAATTCAGGCGGCTCATAGGGC-3′ (SEQ IDNO: 13); PCR was performed as described in Example 4. Followingelectrophoresis through a 2% agarose gel, the 319 bp fragment waspurified by binding to glass beads (Vogelstein et al., Proc. Nat'l.Acad. Sci. USA, Vol. 76, p. 615 (1979)). The DNA fragments were cleavedwith StyI at nt 477 and end-labeled by fill-in with the Klenow fragmentof DNA Polymerase I and ³²P-dCTP. Following electrophoresis of thereaction mixture through a non-denaturing polyacrylamide gel, the 282 bpStyI fragment (nt 477-758), labeled at the proximal end and containingcodon 175, was eluted and purified by extraction with phenol andchloroform. A portion of the eluted DNA was cleaved with HhaI and thefragments separated by electrophoresis on a 6% sequencing gel. Thepresumptive mutation abolished the HhaI site normally present at codon175 (GCGC at nt 522 to 525). Thus, HhaI cleavage of the PCR productsfrom DNA of the normal cells of the patient providing Cx1 (FIG. 5, lanes3 and 4) or from the tumor of another patient (lanes 5 and 6) producedonly the 48 bp product expected if codon 175 was wild-type. In contrast,the PCR product from tumor Cx1 was not cleaved at nt 524 (correspondingto codon 175) and exhibited only a larger 66 bp fragment resulting fromcleavage at a normal downstream HhaI site at nt 542. Analysis of the PCRproduct from paraffin embedded samples of the primary tumor and livermetastasis also exhibited the diagnostic 66 bp HhaI fragment indicatingthe presence of a mutation.

Example 6

This example shows that five out of twenty-one carcinomas tested withthe RNase protection method produced mRNA molecules with detectablesequence mismatches to the wild-type p53 RNA sequence.

Hybrids between a p53 anti-sense RNA probe and p53 mRNA should becleaved by RNase A only at sequence mismatches. Although this method isnot as definitive or as sensitive as sequencing, it allows rapidscreening of a larger number of tumors. Twenty-one colorectal carcinomas(6 primary tumors and 15 cell lines) were examined with probes thatincluded most of the p53 coding region.

Ten ug of cellular RNA was hybridized with radiolabeled anti-sense p53RNA probe, and the hybrids digested with RNase A. A ³²P-labelled RNAprobe was generated in vitro from a p53 cDNA subclone in Bluescript(Stratagene Cloning Systems, La Jolla, Calif.). The probe included 561nt of p53 mRNA coding sequence (nt 473-1034 relative to the translationstart site) plus 60 nt derived from the vector.

The protected fragments were separated by electrophoresis throughdenaturing polyacrylamide gels; autoradiographs of the gels arepresented in FIG. 6. The RNA was derived from: lane 1: S115, carcinomabiopsy; lane 2: SW1417; lane 3: SW948; lane 4: RKO; lane 5: SW480; lane6: RCA; lane 7: GEO; lane 8: FET, lane 9: xenograft Cx3; lane 10: normalcolonic mucosa; lane 11: yeast tRNA; lane 12: probe alone (not digestedwith RNase A); lane 13: SW1417 (long exposure). The fragments markedwith arrowheads in lanes 5, 6 and 13 were not present in the othersamples. The autoradiographic exposure time for lane 13 was 72 hours toallow adequate visualization of the new fragments; for all other lanesthe exposure time was 10 hours.

The RNA from S carcinomas protected fragments of a different size thanthose seen with RNA from normal cells. In two cases, the new fragmentswere the major fragments detected (FIG. 6, lanes 6 and 13, arrowheads).In other cases, the new fragments were of minor intensity compared tothe fully protected fragment (for example, SW480 in lane 5).

Such partial cleavages are not unexpected; the mutations in Cx3 and Cx1were not detected by the RNase protection method (data not shown) and itis known that the majority of RNA sequence mismatches are partially ortotally resistant to RNaseA.

Using similar techniques, five additional colorectal cancers, two breasttumors and one lung tumor have been examined for p53 gene mutations. Inall cases, point mutations of the p53 gene were observed.

Example 7

This example demonstrates that a variety of types of tumors exhibitmutations in the p53 gene; that most tumors with allelic deletions ofp53 have a mutation in the retained allele; that even some tumors withno p53 deletion have mutations in the p53 gene; and that the p53mutations are clustered in four hot-spots on the gene.

A Variety of Tumors Carry p53 Mutations

We analyzed p53 sequences of tumors derived from the breast, lung,brain, colon, or mesenchyme. Tumors of these types have been previouslyshown to exhibit frequent deletions of chromosome 17p when studied byrestriction fragment length polymorphism (RFLP) methods. To test forallelic deletions, tumor DNA samples were digested with HinfI and,following Southern transfer, hybridized sequentially to two probes(p144D6 (Kondoleon, et al., Nucleic Acids Res., vol. 15, p. 10605, 1987)and pYNZ22.1 (Nakamura, et al., Nucleic Acids Res., vol. 16, p. 5707,1988)) detecting variable number of tandem repeat (“VNTR” or“mini-satellite”) sequences. DNA samples from normal tissues exhibitedtwo alleles with at least one of these probes in 29 of 31 differentindividuals tested. Because of this high degree of polymorphism, allelicloss could be assessed with greater than 95% certainty in cell lines andxenografts even when corresponding normal tissue was not available forcomparison.

Nineteen tumors with allelic deletions of chromosome 17p were selectedfor sequence analysis. For tumor cell lines and for xenografts passagedin athymic nude mice, cDNA was generated from mRNA using oligo dT as aprimer. A 1300 bp fragment including the entire p53 coding region wasgenerated from the cDNA using PCR, and this fragment was cloned andsequenced in its entirety. For primary tumors, sufficient RNA was oftennot available for the first approach, and PCR was used to generate a 2.9kb fragment from tumor DNA. This was the longest fragment that we couldreproducibly amplify from the p53 locus, and included all of the exonsfound to contain mutations through the first approach.

RNA was purified using guanidinium isothiocyanate (Chomczynski, et al.,Analytical Biochem., vol. 162, p. 156, 1987) and mRNA selected bybinding to Messenger Affinity Paper (Amersham). cDNA was synthesizedfrom 500-750 ng of mRNA using oligo dT as a primer. The oligo dT primerwas removed by isopropanol precipitation; 10 μg of tRNA and sodiumperchlorate (to a final aqueous concentration of 0.5M) were added to thereaction, and this was followed by addition of ½ volume of isopropanol(Kinzler, et al, Nucleic Acids Res., vol. 17, p. 3645, 1989; Haymerle,et al., Nucleic Acids Res., vol. 14, p. 8615, 1986). The cDNA waspelleted by centrifugation for 15 min. at room temperature and used in a50 μl PCR reaction consisting of 35 cycles of 93° (1 minute), W 580 (1minute), and 70° (2 minutes). Two μg of genomic DNA was used in a 200 μlPCR reaction consisting of 30 cycles at 95°. (1 minute), 58° (1 minute),and 70° (4 minutes). PCR reactions contained magnesium chloride at afinal concentration of 2 mM. The primers used were P1:5′-GGAATTCCACGACGGTGACACG-3′ (SEQ ID NO:1); P2:5′-GGAATTCAAAATGGCAGGGGAGGG-3′ (SEQ ID NO: 1 0); P3:5′-GTAGGAATTCGTCCCAAGCAATGGATGAT-3′ (SEQ ID NO:14); P4:5′-CATCGAATTCTGGAAACTTTCCACTTGAT-3′ (SEQ ID NO: 15). All primers hadextraneous nucleotides comprising EcoRI sites at their 5′ ends tofacilitate cloning. The PCR products were digest with EcoRI,fractionated by electrophoresis, and following purification fromagarose, ligated to EcoRI digested Bluescript vectors (Stratagene).Individual clones were sequenced with primers derived from the p53coding and intron sequences (Buchman, et al., Gene, vol. 70, p. 245,1988) using T7 polymerase and the TDMN sequencing method described inDel Sal, et al., Biotech., vol. 7, p. 514, 1989.

Thirteen of the tumors were found to contain a single missense mutation;two tumors each contained two mutations; one tumor contained aframe-shift mutation at codon 293; and no mutation was detected in fourtumors (Table 1). The PCR reaction is known to be associated with arelatively high rate of base misincorporation (Saiki, et al. Science,vol. 239, p. 487, 1988), and we confirmed this observation by notingseveral sequence variants (13 out of 34,000 bp sequenced) in individualclones that were not reproducibly present in other PCR reactions fromthe same tumor sample. All of the mutations listed in Table 1 wereconfirmed by performing a second PCR reaction and re-sequencing theproducts en masse as described below.

TABLE 1 P53 GENE MUTATIONS IN HUMAN TUMORS MUTATION Tumor Tumor TumorCells # of 17p Codon Nucleo-Amino Tumor # Name Type^(a) Tested^(b)Alleles^(c) tide Acid 1 D263 BRAIN B,X 1 175 GCG—CAC Arg—His 2 D274BRAIN X 1 273 GCT—TGT Arg—Cys 3 D303 BRAIN B,X 1 216 GTG—ATG Val—Met 4D317 BRAIN B,X 1 272 GTG—ATG Val—Met 5 D247 BRAIN C 1 NONE DETECTED 6MDA 468 BREAST C 1 273 CGT—CAT Arg—His 7 T470 BREAST C 1 194 CTT—TTTLeu—Phe 8 BT123 BREAST B 1 NONE DETECTED 9 1012 LUNG B 1 293 DELETED a GFrameshift 10 5855 LUNG B 1 NONE DETECTED 11 H231 LUNG C 2 134 TTT—TTAPhe—Leu 12 88-3/14 NFS B,C 1 179 CAT—TAT His—Tyr 13 Cx4A COLON B,X 1 239AAC—AGC Asn—Ser 14 Cx5A COLON X 1 248 CGG—TGG Arg—Trp 15 Cx6A COLON X 1132 AAG—AAC Lys—Asn 133 ATG—TTG Met—Leu 16 Cx7A COLON B,X 2 281 GAC—GGCAsp—Gly 17 CX19A COLON X 2 NONE DETECTED 18 Cx20A COLON B,X 1 175CGC—CAC Arg—His 19 Cx22A COLON X 1 175 CGC—CAC Arg—His 20 Cx26A COLON X1 141 TGC—TAC Cys—Tyr 21 SW480 COLON C 1 273 CGT—CAT Arg—His 309 CCC—TCCPro—Ser 22 SW837 COLON C 1 248 CGG—TGG Arg—Trp ^(a)The brain tumors wereglioblastoma multiforme; the colon and breast tumors wereadenocarcinomas, the NFS tumor was a neurofibrosarcoma developing in apatient with type I neurofibromatosis; H231 was a small cell carcinomaof the lung, and the other two lung tumors were non-small cellcarcinomas. ^(b)B = tumor biopsy; C = cell line passage in vitro; X =xenograft derived from biopsy, passaged in athymic nude mice. Whenevertwo sources of tumor cells are listed, both contained the indicatedmutation. ^(c)The number of alleles was determined by RFLP analysis asdescribed in the text.

Six p53 Mutations are Somatic Mutations

Two observations indicated that the nucleotide substitutions describedin Table I represented somatic mutations. First, none of thesepresumptive mutations have been observed in the sequences of human p53genes derived from normal cells, SV40 transformed fibroblasts, orlymphoblastoid cell lines (Zakut-Houri, et al., EMBO, vol. 4, p. 1251,1985; Lamb, et al., Mol. Cell. Biol., vol. 6, p. 1379, 1986;Matlashewski, Mol. Cell Biol., vol. 7, p. 961, 1987; Harris, et al.,Mol. Cell. Biol., vol. 6, p. 4650, 1986; Matlashewski, et al., EMBO J.,vol. 3, p. 3257, 1984 and our unpublished data). Second, in 6 cases(tumors #2, 3, 9, 12, 13, 16), normal tissue from the patients whosetumors are described in Table I were available for study. To test forthe presence of the presumptive mutations (in the heterozygous state) inthe germline of these patients, a strategy was devised which employedboth PCR and cloning. Although direct sequencing of PCR products hasbeen shown to be possible by several methods, we found that none of thepublished methods could be reproducibly applied to all parts of the p53coding region. To circumvent this difficulty, we cloned the PCR productsinto a phagemid vector and used the DNA pooled from 103 to 104independent phage clones as a template for DNA sequencing.

PCR reactions were carried out as described above and the reactionproducts digested with EcoRI. The entire reaction was ligated to 0.25 μlof lambda ZAP phage vector arms (Stratagene) and packaged using ¼ of aGIGA-PACK extract (Stratagene). E. coli BB4 cells were then infected,and 10³-10⁴ phage clones plated on a 7 cm petri dish. The lambda ZAPvector contains the sequences for a phagemid into which the PCR insertswere cloned, and single stranded DNA phage can be rescued from thelambda phage clones using a helper phage (Short, et al., Nucleic AcidsRes., vol. 16, p. 7583, 1988). An overnight culture of XL-I Blue cells(Stratagene) was grown in 0.4% maltose and resuspended in 1.5 volumes of10 mM magnesium sulfate. Phages were eluted from the 7 cm dish in 5 mlphage dilution buffer (100 mM sodium chloride, 10 mM magnesium sulfate,20 mM Tris, ph 7.5, 0.02% gelatin) for 2 hours at room temperature withgentle agitation. Fifty μl of eluate was used to infect 200 μl of XL-IBlue cells (Stratagene) in the presence of 1 μl helper phage R408 (10¹¹PFU/ml). After 15 min. at 37°, 5 ml of 2×YT broth was added and theculture shaken for 3 hours at 37°, then heated to 70° for 20 min. Celldebris was pelleted at 3000 g for 5 min., and 10 μl of the supernatant,containing single-stranded DNA phage, was used to infect 200 μl of XL-1Blue cells prepared as described above. After 15 min. at 37°, 100 μl ofthe mixture (containing over 10⁴ clones determined by titration on XL-1Blue cells) was inoculated into 50 ml L-Broth and shaken overnight at37°. Double-stranded DNA was isolated by a rapid alkaline lysistechnique (Bimboim, et al., Nucleic Acids Res., vol. 7, p. 1513, 1979)and sequenced as described above. The primer used for sequencing inpanels 1 and 2 was 5′-GAGGCAAGCAGAGGTGG-3′ (SEQ ID NO: 16). The primerused for sequencing in panels 3 and 4 was 5′-TGGTAATCTACTGGGACG-3′ (SEQID NO: 17).

This procedure resulted in sequence data quality as high as thatproduced using individual plasmid DNA clones as templates, and was usedto demonstrate that in each of the six cases noted above, the mutationsin the tumor DNA were not present in the germline of the patient(examples in FIG. 8).

Two Tumors With No Allelic Loss of p53 Carried p53 Mutations

The data described above indicated that most tumors with one 17p allelecontained a mutation of the p53 gene in the remaining allele. To beginto assess the status of tumors which had not lost a 17p allele, weexamined cDNA clones from three such tumors. In each case, two cDNAclones derived from PCR products, generated as described above weresequenced. In one case (tumor #11), both clones contained a single pointmutation at codon 134 (Table I). In the second case (tumor #16), oneclone contained a point mutation at codon 281 and one clone was wildtype. In the third case (tumor #17), both clones were wild type. Toassess the relative expression levels of the mutant alleles, thesequencing strategy employing pooled phage clones was utilized with cDNAfrom tumor mRNA as a template. In tumor number #11, only the mutantallele was expressed (data not shown); in tumor #16, the mutant and wildtype alleles were expressed at approximately equal levels (FIG. 8, Panel4).

The p53 Mutations are Clustered Along the Gene

Altogether, 20 point mutations (19 missense, 1 frameshift) wereidentified in the present example. These are mapped in FIG. 9, togetherwith the two human p53 gene missense mutations previously described(Baker, et al., Science, vol. 244, p. 217, 1989). Several features arenotable. Although the sample size is limited, the mutations tended to beclustered in four hotspots which accounted for 86% of the 21 missensemutations (5 mutations in region A, codons 132-143; five mutations inregion B, codons 174-179; 3 mutations in region C, codons 236-248; 5mutations in region D, codons 272-281). There have been two missensemutations identified in murine tumor cells, both in thecarcinogen-induced fibrosarcoma cell line Meth A: one allele contained amutation in region A, and the other contained one mutation in region Cand one mutation in region D (Finlay, et al., Mol. Cell. Biol., vol. 8,p. 531, 1988; Eliyahu, et al., Oncogene, vol. 3, p. 313, 1988).Interestingly, the four hotspots for in vivo mutation coincided exactlywith the four most highly conserved regions of the p53 gene, previouslyidentified (Soussi, et al., Oncogene, vol. 1, p. 71, 1987). Of the 41amino acids contained within regions A-D, 93% are identical in thewild-type p53 genes of amphibian, avian, and mammalian species, comparedto a conservation of only 51-57% over the entire p53 coding sequence.The clustering of mutations and evolutionary conservation of regions A-Dsuggest that they play a particularly important role in mediating thenormal function of the p53 gene product.

Example 8

This example shows that expression of the wild-type p53 gene in humancolorectal carcinoma cells dramatically inhibits their growth and that amutant p53 gene cloned from a human colorectal carcinoma was incapableof exerting such inhibition.

The colorectal carcinoma lines SW480 and SW837, which are representativeof 75% of colon carcinomas, have each lost one copy of chromosome 17p(including the p53 gene) and the remaining p53 allele is mutated (Baker,et al., Science 244, 217 (1989); Nigro et al, Nature 342, 705 (1989)).The SW837 line contains an arginine to tryptophan mutation at codon 248(Nigro, supra). The SW480 line contains two point mutations, arginine tohistidine at codon 273 and proline to serine at codon 309 (Nigro,supra.). The substitutions at codon 248 and 273 are typical of thoseobserved in human tumors, occurring within two of the four mutation “hotspots” (Nigro, supra).

For the transfection studies, we constructed a vector, pCMV-Neo-Bam,engineered to contain two independent transcription units. Theexpression vector pCMV-Neo-Bam was derived from plasmid BCMGNeo-mIL2(Karasuyama, et al., J. Exp. Med. 169, 13 (1989) by excision of thehuman beta globin sequences and bovine papilloma virus sequences withBamHI and NotI. Next, the interleukin 2 (IL-2) sequences present at theunique XhoI site were removed, and the XhoI site was changed to a BamHIsite by linker addition. The vector included CMV promoter/enhancersequences, which could drive expression of the insert at the BamHI site,and splicing and polyadenylation sites derived from the rabbit betaglobin gene, which ensured proper processing of the transcribed insertin the cells. A pBR322 origin of replication and b-lactamase genefacilitated growth of the plasmid in E. coli. The plasmid conferredgeneticin resistance through expression of the neomycin resistance geneunder separate control of an HSV thymidine kinase promoter. The firsttranscription unit comprised a cytomegalovirus (CMV) promoter/enhancerupstream of a site for insertion of the cDNA sequences to be expressed,and splice and polyadenylation sites to ensure appropriate processing.The second transcription unit included a herpes simplex virus (HSV)thymidine kinase promoter/enhancer upstream of the neomycin resistancegene, allowing for selection of transfected cells in geneticin.

A wild-type p53 cDNA was inserted into pCMV-Neo-Bam to produce pC53-SN3.Similarly, a vector, pC53-SCX3, expressing a mutant cDNA from humancolorectal tumor CX3, was also constructed. The only difference betweenpC53-SN3 and pC53-SCX3 was a single nucleotide (C to T) resulting in asubstitution of alanine for valine at p53 codon 143 in pC53-SCX3. Thetwo constructs were made as follows: a 1.8-kb XbaI fragment, extendingfrom nucleotide −130 to 1671 relative to the translation initiationsite, was isolated from wild-type or CX3 cDNA clones. The fragment wasblunt-ended with the Klenow fragment of DNA polymerase, ligated to BamHIlinkers, and cloned into the unique BamHI site in the expression vectorpCMV-Neo-Bam.

The constructs were transfected into SW837 and SW480 cells (obtainedfrom the American Type Culture Collection, Rockville, Maryland), andgeneticin-resistant colonies were counted 3 weeks later. Cellstransfected with pC53-SN3 formed five-to tenfold fewer colonies thanthose transfected with pC53-SCX3 in both recipient cell types (Table 2).

TABLE 2 Colony formation after transfection with wild-type and mutantp53 expression vectors. For each experiment, one or two 75-cm² flaskswere transfected, and the total colonies counted after 3 to 4 weeks ofselection in geneticin (0.8 mg/ml). Exp. experiment. No. ofgeneticin-resistant colonies formed Cell pC53-SCX3 pC53-SN3 line Exp.(mutant) (wild-type) SW837 1  754  66 2  817  62 SW480 1 449  79 2 364 26 RKO 1 1858 190 2 1825 166 VACO 235 1  18  16 2  26  28

In both SW837 and SW480 cells, the number of colonies produced by theexpression vector pCMV-Neo-Bam (without a p53 cDNA insert) was similarto that induced by the pC53-SCX3 construct.

These results suggest that the wild-type p53 gene inhibits the clonalgrowth of both the SW837 and SW480 cell lines; however, a significantnumber of colonies formed after transfection of the wild-type construct.If wild-type p53 expression were truly inhibitory to cell growth, onewould expect that no colonies would form or that p53 expression in thecolonies that did form would be reduced compared to that produced withthe mutant p53 cDNA construct. To evaluate this issue, we expandedindependent SW480 and SW837 colonies into lines, and ribonuclease(RNase) protection analysis was performed to determine the amount of p53mRNA expressed from the exogenously introduced sequences. Twelve of 31lines (38%) derived from transfection with the pC53-SCX3 construct werefound to express the exogenous mutant p53 mRNA. This percentage wasconsistent with results expected in human cells transfected with avector containing two independent transcription units. Previous studieshave shown that, in contrast to rodent cells, primate cells are able tointegrate only a small amount of foreign DNA (approximately 6 kb), sothat only 10 to 30% of clones selected for the expression of onetranscription unit also contain the second unit in an intact form (F.Collabere-Garapin, et al., Gene 50, 279 (1986); Hoeijmakers, et al.,Exp. Cell Res., 169, 111 (1987); Mayne et al., Gene 66, 65 (1988), Dean,et al., Exp. Cell Res., 183, 473 (1989). In contrast, no expression ofexogenous p53 wild-type mRNA was seen in any of 21 clonal linesestablished from either SW480 or SW837 cells transfected with thepC53-SN3 vector (FIG. 10A). These RNase protection results weresupported by analysis of the exogenous p53 DNA sequences within theclones. All of the p53-expressing clones derived from the pC53-SCX3transfection contained an intact copy of the exogenous p53 gene (FIG.10B). In contrast, in all the clones derived from the pC53-SN3transfection, the exogenous p53 sequences were deleted or rearranged(FIG. 10B).

The results from individual clones were further supported by theanalysis of pooled clones, in which numerous colonies could besimultaneously assessed. Forty or more clones from two to three separatetransfection experiments were pooled and analyzed approximately 3 weeksafter transfection. RNase protection studies showed substantialexpression of wild-type sequences was not detectable (FIG. 11A). Resultsfrom Southern (DNA) blotting were consistent with the RNase protectionstudies, in that pooled colonies from the wild-type transfectants had nodetectable unrearranged exogenous p53 sequences, in contrast to theintact p53 sequences in colonies derived from the mutant p53 cDNAexpression vector (FIG. 11B).

The conclusions made from the above experiments are dependent on theassumption that p53 protein was produced in the transfected cell lines.Clones containing exogenous mutant p53 sequences produced p53 mRNA at aconcentration 1.5 to 3.5 time higher than that produced by theendogenous p53 gene (FIGS. 10A and 11A). Immunoblot analysis showed thatthere was a concomitant small increase in p53 protein expression in thetransfectants (1.5- to 3-fold) compared to the untransfected cells.However, this increase was difficult to measure quantitatively, sincethese cells produced significant amounts of endogenous p53 protein that(unlike endogenous p53 mRNA) could not be distinguished from thatproduced by the vectors. To confirm that transfected human cellsexpressed p53 protein from our constructs, we studied an additionalcolorectal carcinoma cell line (RKO). RKO cells were obtained throughthe generosity of M. Brattain. Although RKO cells did not contain amutation within the susceptible p53 coding sequences, i.e., exons 5-9,they expressed low concentrations of p53 mRNA compared to normalcolorectal mucosa or the other lines studied and did not producedetectable amounts of protein.

Results of colony formation assays in transfected RKO cells were similarto those in SW480 and SW837 cells. Colony formation by wild-type p53gene transfectants occurred with a tenfold decrease in efficiencycompared to the mutant p53 construct (Table 2). Immunocytochemicaldetection of p53 protein in transfected RKO cells was done as follows:approximately 5×10⁴ cells were cytocentrifuged onto polylysine-coatedslides, fixed for 10 min in formalin, and permeabilized for 5 min in0.5% Triton X-100. A mouse monoclonal antibody against human p53 protein(Ab1801) in combination with the ABC immunoperoxidase system (VectorLaboratories), was used for immunocytochemical detection of p53 protein(Banks, et al., Eur. J. Biochem. 159, 529 (1986)). Ten to 20 randomlyselected microscopic fields were analyzed per slide. These observationsare consistent with the greater stability of mutant compared towild-type p53 protein noted previously (C. A. Finlay et al., Mol. CellBiol. 8, 531 (1988)). However, transient mRNA expression was alsosignificantly lower in the SN3 transfectants compared to the SCX3transfectants at 48 and 96 hours, supporting the idea that RKO cellsexpressing wild-type p53 were at a selective disadvantage compared tothose producing mutant p53 products.

To obtain additional evidence that cells expressing p53 were inhibitedin their growth potential, we examined the effect of p53 gene expressionon DNA synthesis in transfected RKO cells were labeled with[³H]thymidine for 2 hours. The cells were subsequently fixed,immunocytochemically stained for the presence of p53 protein, andautoradiographed. The number of cells undergoing DNA replication wasonly slightly lower in cells producing exogenous mutant p53 protein thanin cells that did not express any detectable p53 protein. Expression ofthe wild-type protein, however, dramatically inhibited the incorporationof thymidine (Table 3).

TABLE 3 Immunocytochemistry and [³H]thymidine incorporation oftransfected RKO cells. Percent of cells Percent of cells expressing p53incorporating protein at [³H]thymidine in 6 24 48 96 p53 p53 non-Plasmid hrs hrs hrs hrs expressors expressors pC53-SCX3 1.0 11 4.3 2.024 31 pC53-SN3 1.9 5.2 0.3 0.2 1.7 33

These results all suggested that wild-type p53 exerted an inhibitoryeffect on the growth of carcinoma cells in vitro. To evaluate whetherthis inhibitory effect was cell type-specific, we transfected colorectalepithelial cells derived from a benign tumor of the colon (the VACO 235adenoma cell line). VACO 235 cells are described by J. K. V. Willson etal., Cancer Res., 47, 2704 (1987). Previous studies have shown that mostadenomas contain two copies of chromosome 17p and express wild-type p53mRNA at concentrations similar to that of normal colonic mucosa.Analogously, the p53 alleles of the VACO 235 cell line were sequenced(exons 5-9) and found to be wild-type and the expression of p53 mRNA wasfound to be similar to that of normal colorectal mucosa. In contrast tothe results seen with SW480, SW837, and RKO cells, the pC53-SN3 andpC53-SCX3 constructs produced similar numbers of geneticin-resistantcolonies after transfection of the VACO 235 line (Table 2). Weconsidered, however, that the most definitive test for differentialgrowth inhibition by wild-type versus mutant p53 genes involved analysisof exogenous p53 expression in pooled transfectants. Through suchanalysis, a large number of colonies could be examined simultaneouslyand the expression of exogenous mutant and wild-type p53 genes directlycompared. Striking differences in the relative expression from thetransfected genes were seen in all three carcinoma cell lines tested.VACO 235 transfectants, however, expressed similar amounts of exogenousp53 mRNA from either pC53-SN3 (wild-type) or pC53-SCX3 (mutant) p53constructs (FIG. 11A).

In summary, our results suggest that expression of the wild-type p53gene in colorectal carcinoma cell lines was incompatible withproliferation. The inhibitory effects of wild-type p53 were specific intwo ways. First, a single point mutation in a p53 gene constructabrogated its suppressive properties as measured by three separateassays (colony formation, exogenous p53 expression in transfectedclones, and thymidine incorporation). The CX3 mutant provided a controlfor gene specificity as it contained only one conservative mutation,resulting in a substitution of one hydrophobic amino acid (alanine) foranother (valine) at a single codon. Second, the growth-suppressiveeffect of the wild-type p53 construct was cell type-specific.Introduction of the wild-type vector into the VACO 235 adenoma cell linehad no measurable inhibitory effect compared to the mutant p53 vector.There are several differences between the cell lines that could accountfor the differential effect of the introduced vectors. Regardless of thebasis for the difference, the results with the VACO 235 cell lineminimize the possibility that the wild-type p53 construct had somenonspecific, toxic effect on recipient cells; the effect was celltype-dependent.

The transfection and expression results of Table 2 and FIG. 11A suggestthat cells at the premalignant stages of tumor progression (VACO 235)may be less sensitive to the inhibitory effects of wild-type p53 thanmalignant cells (SW480, SW837, and RKO). This hypothesis is consistentwith previous results that suggest the wild-type p53 is less inhibitoryto the growth of normal rat embryo fibroblasts than to theironcogene-transfected derivatives. Finlay et al., Cell 57, 1083 (1989);Eliyahu et al., Proc. Natl. Acad. Sci. USA, 86, 8763 (1989). Thissensitivity may only be relative: expression of the wild-type gene athigh concentrations might inhibit the growth of any cell type, includingnon-neoplastic cells, by overwhelming normal regulatory processes suchas phosphorylation. Samad et al., Proc. Natl. Acad. Sci. USA, 83, 897(1986); Meek et al., Mol. Cell Biol. 8, 461 (1988). Genetic alterationsthat occur during the progression of colorectal tumors may increase thesensitivity of cells to p53 inhibition, making wild-type p53 expressiona key, rate-limiting factor for further tumor growth and expansion. Atthis point, and not before, mutations in the p53 gene would confer aselective growth advantage to cells in vivo, which would explain thefrequent occurrence of p53 gene mutations and allelic loss only in themore advanced stages of colorectal tumorigenesis.

17 1 22 DNA Homo sapiens 1 ggaattccac gacggtgaca cg 22 2 24 DNA Homosapiens 2 ggaattcggt gtaggagctg ctgg 24 3 23 DNA Homo sapiens 3ggaattccca gaatgccaga ggc 23 4 25 DNA Homo sapiens 4 ggaattcatgtgctgtgact gcttg 25 5 21 DNA Homo sapiens 5 ggaattccac acccccgccc g 21 622 DNA Homo sapiens 6 ggaattcatg ccgcccatgc ag 22 7 24 DNA Homo sapiens7 ggaattctga ctgtaccacc atcc 24 8 23 DNA Homo sapiens 8 ggaattctccatccagtggt ttc 23 9 24 DNA Homo sapiens 9 ggaattccca acaacaccag ctcc 2410 23 DNA Homo sapiens 10 gaattcaaaa tggcagggga ggg 23 11 21 DNA Homosapiens 11 ttcctcttcc tgcagtactc c 21 12 18 DNA Homo sapiens 12gacgcgggtg ccgggcgg 18 13 24 DNA Homo sapiens 13 cggaattcag gcggctcatagggc 24 14 29 DNA Homo sapiens 14 gtaggaattc gtcccaagca atggatgat 29 1529 DNA Homo sapiens 15 catcgaattc tggaaacttt ccacttgat 29 16 17 DNA Homosapiens 16 gaggcaagca gaggtgg 17 17 18 DNA Homo sapiens 17 tggtaatctactgggacg 18

What is claimed is:
 1. A method of supplying wild-type p53 gene functionto a cell which has lost said gene function by virtue of a mutation in ap53 gene, comprising: introducing a portion of a human wild-type p53gene into a human cell which has lost said gene function such that saidportion is expressed in the cell, said portion encoding a part of humanwild-type p53 protein which is required for non-neoplastic growth ofsaid cell, whereby wild-type p53 gene function is supplied to the cell.2. The method of claim 1 wherein said portion corresponds to a region ofthe p53 gene in the cell which contains the mutation.